The NG2 proteoglycan is not expressed by mammary tumor cells in the MMTV-PyMT mouse model of breast cancer, but is expressed by at least three important cell types in the tumor stroma: pericytes in the tumor vasculature, myeloid cells that invade the tumors from the circulation, and adipocytes in the mammary fat pad. By several criteria, including tumor latency, growth rate, and metastasis, ablation of NG2 greatly slows mammary tumor progression in the MMTV-PyMT model, emphasizing the power of microenvironmental factors in promoting tumorigenesis. Due to our interest in tumor vascularization and metastasis, we are focusing on mechanisms by which NG2 supports the tumor-promoting activities of pericytes and myeloid cells. The specific aims of this proposal will be to examine the respective effects of pericyte NG2 and myeloid cell NG2 on mammary tumor progression in the MMTV-PyMT model. For these purposes we will utilize cell type-specific ablations of NG2 in these two populations to analyze the progression of both spontaneous and allografted mammary tumors. In Aim 1 we will compare tumor progression and tumor vascularization in control mice and in pericyte-specific NG2 null mice produced by crossing NG2 floxed mice with Pdgfrb/Cre transgenic mice. Characterization of the tumor vasculature will include determinations of pericyte ensheathment of endothelial cells, maturation of pericytes and endothelial cells, assembly of the basal lamina, vessel patency, vessel leakiness, and tumor hypoxia. In vitro co-cultures of pericytes and endothelial cells will be used to further elucidate mechanisms by which NG2 supports pericyte/endothelial cell crosstalk. In Aim 2 we will compare myeloid cell function in control mice and in myeloid-specific NG2 null mice produced by crossing NG2 floxed mice with LysM/Cre transgenic mice. Characterization of the effects of NG2 ablation will include determination of M1 versus M2 polarization, assessment of changes in the size and differentiation state of key myeloid populations, and localization of these populations to critical sites such as vasculature and tumor margins. In vitro co-cultures of tumor cells and macrophages will be used to explore mechanisms by which NG2 affects macrophage/tumor cell interaction. In Aim 3 we will use both the pericyte-specific and myeloid-specific NG2 null mice to study the importance of NG2 in mammary tumor metastasis to the lungs. We will use fluorescent-labeled mammary tumor cells to dissect the metastatic process into its component stages, including intravasation of tumor cells into the vasculature, extravasation of tumor cells from the vasculature into the lungs, and establishment of pre-metastatic niches in the lungs.